Small ubiquitin-related modifier (SUMO)-1 promotes glycolysis in hypoxia.

Under conditions of hypoxia, most eukaryotic cells undergo a shift in metabolic strategy, which involves increased flux through the glycolytic pathway. Although this is critical for bioenergetic homeostasis, the underlying mechanisms have remained incompletely understood. Here, we report that the induction of hypoxia-induced glycolysis is retained in cells when gene transcription or protein synthesis are inhibited suggesting the involvement of additional post-translational mechanisms. Post-translational protein modification by the small ubiquitin related modifier-1 (SUMO-1) is induced in hypoxia and mass spectrometric analysis using yeast cells expressing tap-tagged Smt3 (the yeast homolog of mammalian SUMO) revealed hypoxia-dependent modification of a number of key glycolytic enzymes. Overexpression of SUMO-1 in mammalian cancer cells resulted in increased hypoxia-induced glycolysis and resistance to hypoxia-dependent ATP depletion. Supporting this, non-transformed cells also demonstrated increased glucose uptake upon SUMO-1 overexpression. Conversely, cells overexpressing the de-SUMOylating enzyme SENP-2 failed to demonstrate hypoxia-induced glycolysis. SUMO-1 overexpressing cells demonstrated focal clustering of glycolytic enzymes in response to hypoxia leading us to hypothesize a role for SUMOylation in promoting spatial re-organization of the glycolytic pathway. In summary, we hypothesize that SUMO modification of key metabolic enzymes plays an important role in shifting cellular metabolic strategies toward increased flux through the glycolytic pathway during periods of hypoxic stress.

Ecto-5'-nucleotidase (CD73) regulation by hypoxia-inducible factor-1 mediates permeability changes in intestinal epithelia.

Under conditions of limited oxygen availability (hypoxia), multiple cell types release adenine nucleotides in the form of ATP, ADP, and AMP. Extracellular AMP is metabolized to adenosine by surface-expressed ecto-5'-nucleotidase (CD73) and subsequently activates surface adenosine receptors regulating endothelial and epithelial barrier function. Therefore, we hypothesized that hypoxia transcriptionally regulates CD73 expression. Microarray RNA analysis revealed an increase in CD73 and ecto-apyrase CD39 in hypoxic epithelial cells. Metabolic studies of CD39/CD73 function in intact epithelia revealed that hypoxia enhances CD39/CD73 function as much as 6 +/- 0.5-fold over normoxia. Examination of the CD73 gene promoter identified at least one binding site for hypoxia-inducible factor-1 (HIF-1) and inhibition of HIF-1alpha expression by antisense oligonucleotides resulted in significant inhibition of hypoxia-inducible CD73 expression. Studies using luciferase reporter constructs revealed a significant increase in activity in cells subjected to hypoxia, which was lost in truncated constructs lacking the HIF-1 site. Mutagenesis of the HIF-1alpha binding site resulted in a nearly complete loss of hypoxia-inducibility. In vivo studies in a murine hypoxia model revealed that hypoxia-induced CD73 may serve to protect the epithelial barrier, since the CD73 inhibitor alpha,beta-methylene ADP promotes increased intestinal permeability. These results identify an HIF-1-dependent regulatory pathway for CD73 and indicate the likelihood that CD39/CD73 protects the epithelial barrier during hypoxia.

Hypoxic regulation of NF-kappaB signaling.

Hypoxia and inflammation are coincidental events in an array of diseased tissues, including chronically inflamed sites (e.g., inflammatory bowel disease, rheumatoid arthritis), growing tumors, myocardial infarcts, atherosclerotic plaques, healing wounds, and sites of bacterial infection (Murdoch et al., 2005). An understanding of how hypoxia modulates the inflammatory response is critical in developing our fundamental understanding of inflammatory disease and identifying new windows of therapeutic opportunity. Nuclear factor-kappaB (NF-kappaB) is a master transcriptional regulator of inflammatory and antiapoptotic gene expression, the activation of which has significant implications in disease development. Recent work has uncovered mechanisms by which hypoxia modulates the activation of NF-kappaB in cells through decreased oxygen-dependent suppression of the key regulators of this pathway. This work has implicated a novel role for proline and asparagine hydroxylases in the modulation of NF-kappaB activity. Here, we describe methodologies used to demonstrate and interrogate hypoxic induction of the NF-kappaB pathway.

c-Jun NH2-terminal kinase activation contributes to hypoxia-inducible factor 1alpha-dependent P-glycoprotein expression in hypoxia.

We previously have shown that hypoxia increases the expression of P-glycoprotein, which in turn increases tumor cell capacity to actively extrude chemotherapeutic agents and may contribute to tumor drug resistance. This event is mediated through the hypoxia-inducible factor (HIF-1). Here, we investigated the role of the stress-activated protein kinase c-Jun NH(2)-terminal kinase (JNK) in the signaling mechanisms underlying these events. Hypoxia activates JNK activity in vitro and in vivo. Overexpression of mitogen-activated protein kinase (MAPK) kinase kinase (MEKK-1), which preferentially activates JNK, mimics, in a nonadditive way, hypoxia-induced activity of the MDR1 promoter and expression of MDR1 mRNA and P-glycoprotein. Furthermore, the JNK inhibitor SP600125 selectively and specifically inhibits hypoxia- and MEKK-1-induced MDR1 promoter activity in a dose-dependent manner. JNK inhibition also reversed hypoxia- and MEKK-1-induced activity of an HIF-1-dependent reporter gene. MEKK-1-induced MDR1 expression depends on a functional HIF-1 binding site (hypoxia-responsive element). Hypoxia- but not cobalt chloride-dependent HIF-1-DNA binding and transcriptional activation was inhibited by SP600125, indicating that hypoxia-induced signaling to HIF-1 depends on JNK activation. Because it has been reported that reactive oxygen species are increased in hypoxia and related to JNK activation, we investigated their role in signaling this response. Whereas exogenous addition of H(2)O(2) was sufficient to activate JNK, reactive oxygen species scavengers were without effect on hypoxia-induced JNK or HIF-1 activation. Thus, hypoxia-elicited MDR1 expression, which depends on HIF-1 activation, depends at least in part on signaling via activation of JNK. Furthermore, these events are independent of the generation of reactive oxygen intermediates. Thus, JNK may represent a therapeutic target in the prevention of tumor resistance to chemotherapeutic treatment.

Hypoxia-inducible factor-1-dependent regulation of the multidrug resistance (MDR1) gene.

The microenvironment of rapidly growing tumors is associated with increased energy demand and diminished vascular supply, resulting in focal areas of prominent hypoxia. A number of hypoxia-responsive genes have been associated with growing tumors, and here we demonstrate that the multidrug resistance (MDR1) gene product P-glycoprotein, a Mr approximately 170,000 transmembrane protein associated with tumor resistance to chemotherapeutics, is induced by ambient hypoxia. Initial studies using quantitative microarray analysis of RNA revealed an approximately 7-fold increase in MDR in epithelial cells exposed to hypoxia (pO(2) 20 torr, 18 h). These findings were further confirmed at the mRNA and protein level. P-Glycoprotein function was studied by analysis of verapamil-inhibitable efflux of digoxin and rhodamine 123 in intact T84 cells and revealed that hypoxia enhances P-glycoprotein function by as much as 7 +/- 0.4-fold over normoxia. Subsequent studies confirmed hypoxia-elicited MDR1 gene induction and increased P-glycoprotein expression in nontransformed, primary cultures of human microvascular endothelial cells, and analysis of multicellular spheroids subjected to hypoxia revealed increased resistance to doxorubicin. Examination of the MDR1 gene identified a binding site for hypoxia inducible factor-1 (HIF-1), and inhibition of HIF-1 expression by antisense oligonucleotides resulted in significant inhibition of hypoxia-inducible MDR1 expression and a nearly complete loss of basal MDR1 expression. Studies using luciferase promoter constructs revealed a significant increase in activity in cells subjected to hypoxia, and such hypoxia inducibility was lost in truncated constructs lacking the HIF-1 site and in HIF-1 binding site mutants. Extensions of these studies also identified a role for Sp1 in this hypoxia response. Taken together, these data indicate that the MDR1 gene is hypoxia responsive, and such results may identify hypoxia-elicited P-glycoprotein expression as a pathway for resistance of some tumors to chemotherapeutics.

Role of vasodilator-stimulated phosphoprotein in PKA-induced changes in endothelial junctional permeability.

At sites of ongoing inflammation, polymorphonuclear leukocytes (PMN, neutrophils) migrate across vascular endothelia, and such transmigration has the potential to disturb barrier properties and can result in intravascular fluid loss and edema. It was recently appreciated that endogenous pathways exist to dampen barrier disruption during such episodes and may provide an important anti-inflammatory link. For example, during transmigration, PMN-derived adenosine activates endothelial adenosine receptors and induces a cAMP-dependent resealing of endothelial barrier function. In our study reported here, we sought to understand the link between cyclic nucleotide elevation and increased endothelial barrier function. Initial studies revealed that adenosine-induced barrier function is tightly linked to activation of protein kinase A (PKA). Because PKA selectively phosphorylates serine and threonine residues, we screened zonula occludens-1 (ZO-1) immunoprecipitates for the existence of such phosphorylated proteins as targets for barrier regulation. This analysis revealed a dominantly phosphorylated band at 50 kDa. Microsequencing identified this protein as vasodilator-stimulated phosphoprotein (VASP), an actin binding protein with multiple serine/threonine phosphorylation sites. Immunofluorescent microscopy revealed that VASP localizes to endothelial junctional complexes and colocalizes with ZO-1, occludin, and junctional adhesion molecule-1 (JAM-1). To address the role of phospho-VASP in regulation of barrier function, we generated a phosphospecific VASP antibody targeting the Ser157 residue phosphorylation site, the site preferred by PKA. Immunolocalization studies with this antibody revealed that upon PKA activation, phospho-VASP appears at cell-cell junctions. Transient transfection of truncated VASP fragments revealed a parallel increase in barrier function. Taken together, these studies reveal a central role for phospho-VASP in the coordination of PKA-regulated barrier function, such as occurs during episodes of inflammation.

Epithelial cell-neutrophil interactions in the alimentary tract: a complex dialog in mucosal surveillance and inflammation.

Inflammatory diseases of mucosal organs as diverse as the lung, kidney, and intestine, inevitably require the intimate interactions of neutrophils with columnar epithelia. The physiologic consequences of such interactions often determine endpoint organ function, and for this reason, much recent interest has developed in identifying mechanisms and novel targets for the treatment of mucosal inflammation. Elegant in vitro model systems incorporating purified human neutrophils and human epithelial cells grown in physiologic orientations have aided in discovery of new and insightful pathways to define basic inflammatory pathways. Here, we will review the recent literature regarding the interactions between columnar epithelial cells and neutrophils, with an emphasis on intestinal epithelial cells, structural aspects of neutrophil transepithelial migration, molecular determinants of neutrophil-epithelial cell interactions, as well as modulation of these pathways. These recent studies highlight the dynamic nature of these pathways and lend insight into the complexity of treating mucosal inflammation.

Hypoxia induces epithelial amphiregulin gene expression in a CREB-dependent manner.

Hypoxia occurs during a number of conditions in which altered epithelial proliferation is critical, including tumor development. Microarray analysis of colon-derived epithelial cells revealed a hypoxia-dependent increase in the expression of amphiregulin, an EGF receptor (EGFR) ligand that activates epithelial proliferation and has been associated with the development of colonic tumors. Amphiregulin expression was also induced in tissues from mice exposed to whole animal hypoxia. The hypoxic upregulation of amphiregulin was independent of the classic transcriptional response mediated via hypoxia-inducible factor (HIF)-1alpha. Transfection of HeLa cells with truncated amphiregulin promoter reporter constructs revealed that a 37-bp segment upstream from the TATA box retained hypoxic sensitivity. This sequence contains an evolutionarily conserved cAMP response element (CRE) that constitutively binds the CRE binding protein (CREB). Deletion of the CRE abolished sensitivity to hypoxia. Thus hypoxia promotes intestinal epithelial amphiregulin expression in a CRE-dependent manner, an event that may contribute to increased proliferation. These data also further support a role for CREB as an HIF-independent hypoxia-responsive transcription factor in the regulation of intestinal epithelial gene expression.

Prolyl hydroxylase-1 negatively regulates IkappaB kinase-beta, giving insight into hypoxia-induced NFkappaB activity.

Hypoxia is a feature of the microenvironment of a growing tumor. The transcription factor NFkappaB is activated in hypoxia, an event that has significant implications for tumor progression. Here, we demonstrate that hypoxia activates NFkappaB through a pathway involving activation of IkappaB kinase-beta (IKKbeta) leading to phosphorylation-dependent degradation of IkappaBalpha and liberation of NFkappaB. Furthermore, through increasing the pool and/or activation potential of IKKbeta, hypoxia amplifies cellular sensitivity to stimulation with TNFalpha. Within its activation loop, IKKbeta contains an evolutionarily conserved LxxLAP consensus motif for hydroxylation by prolyl hydroxylases (PHDs). Mimicking hypoxia by treatment of cells with siRNA against PHD-1 or PHD-2 or the pan-prolyl hydroxylase inhibitor DMOG results in NFkappaB activation. Conversely, overexpression of PHD-1 decreases cytokine-stimulated NFkappaB reporter activity, further suggesting a repressive role for PHD-1 in controlling the activity of NFkappaB. Hypoxia increases both the expression and activity of IKKbeta, and site-directed mutagenesis of the proline residue (P191A) of the putative IKKbeta hydroxylation site results in a loss of hypoxic inducibility. Thus, we hypothesize that hypoxia releases repression of NFkappaB activity through decreased PHD-dependent hydroxylation of IKKbeta, an event that may contribute to tumor development and progression through amplification of tumorigenic signaling pathways.

Role of VASP in reestablishment of epithelial tight junction assembly after Ca2+ switch.

Epithelial permeability is tightly regulated by intracellular messengers. Critical to maintaining barrier integrity is the formation of tight junction complexes. A number of signaling pathways have been implicated in tight junction biogenesis; however, the precise molecular mechanisms are not fully understood. A growing body of evidence suggests a role for intracellular cAMP in tight junction assembly. Using an epithelial model, we investigated the role of cAMP signal transduction in barrier recovery after Ca2+ switch. Our data demonstrate that elevation of intracellular cAMP levels significantly enhanced barrier recovery after Ca2+ switch. Parallel experiments revealed that epithelial barrier recovery is diminished by H-89, a specific and potent inhibitor of cAMP-dependent protein kinase (protein kinase A) activity. Of the possible PKA effector proteins, the vasodilator-stimulated phosphoprotein (VASP) is an attractive candidate, since it has been implicated in actin-binding and cross-linking functions. We therefore hypothesized that VASP may play a role in the cAMP-mediated regulation of epithelial junctional reassembly after Ca2+ switch. We demonstrate here that VASP is phosphorylated via a PKA-dependent process under conditions that enhance barrier recovery. Confocal laser scanning microscopy studies revealed that VASP localizes with ZO-1 at the tight junction and at cell-cell borders and that phospho-VASP appears at the junction after Ca2+ switch. Subsequent transfection studies utilizing epithelial cells expressing truncated forms of VASP abnormal in oligomerization or actin-binding activity revealed a functional diminution of barrier recovery after Ca2+ chelation. Our present studies suggest that VASP may provide a link between cAMP signal transduction and epithelial permeability.

Small ubiquitin-related modifier-1 modification mediates resolution of CREB-dependent responses to hypoxia.

Phosphorylation-dependent ubiquitination combined with proteasomal degradation of transcriptional regulators is a recently appreciated mechanism for control of a number of inflammatory genes. Far less is known about the counterregulatory mechanisms that repress transcriptional activity in these pathways during resolution. Here, we investigated the transient nature of hypoxia-induced tumor necrosis factor (TNF)alpha in T84 cells, a process we have previously shown to involve phosphorylation-dependent degradation of the cAMP-response element-binding protein (CREB). Initial studies indicate hypoxia-induced TNFalpha to be a transient event, the resolution of which is associated with the appearance of a higher molecular weight modified form of CREB. Gene array analysis of mRNA derived from hypoxic cells identified a time-dependent induction of small ubiquitin-related modifier (SUMO)-1 mRNA. In prolonged hypoxia, CREB is posttranslationally modified by SUMO-1. Furthermore, SUMO-1 overexpression stabilizes CREB in hypoxia and enhances CREB-dependent reporter gene activity. Site-directed mutagenesis of lysine residues K285 and K304 identifies them as SUMO acceptors in vivo and in vitro. Mutation of K304 also results in loss of CREB nuclear localization, implying a role for SUMO-1 modification at this site in the subcellular localization of CREB. Thus, in prolonged hypoxia, CREB is modified by association with SUMO-1. Furthermore, we hypothesize that such an event stabilizes and promotes nuclear localization of CREB and thus complements an endogenous resolution phase for hypoxia-induced inflammatory processes.